[CANCER RESEARCH 64, 6511–6523, September 15, 2004 ... · cell carcinomas (SCCs) that arise from...

[CANCER RESEARCH 64, 6511–6523, September 15, 2004]

Nuclear Factor-�B is an Important Modulator of the Altered Gene ExpressionProfile and Malignant Phenotype in Squamous Cell Carcinoma

Amy Loercher,1 Tin Lap Lee,1 Justin L. Ricker,1 April Howard,1 Joel Geoghegen,1 Zhong Chen,1 John B. Sunwoo,1

Raquel Sitcheran,3 Eric Y. Chuang,2 James B. Mitchell,2 Albert S. Baldwin, Jr.,3 and Carter Van Waes1,2

1Head and Neck Surgery Branch, National Institute on Deafness and Other Communication Disorders, Rockville, Maryland; 2Radiation Oncology Sciences Program, NationalCancer Institute, Bethesda, Maryland; and 3Lineberger Cancer Center, University of North Carolina, Chapel Hill, North Carolina

ABSTRACT

We reported previously that transcription factor nuclear factor(NF)-�B is constitutively activated in human and murine squamous cellcarcinomas (SCCs). The role of NF-�B in the cumulative changes in geneexpression with transformation and progression of the murine SCC Pam212 and after switching off NF-�B by a dominant negative inhibitor �Bmutant (I�B�M) was explored by profiling with a 15,000-element cDNAmicoarrray. Remarkably, NF-�B modulated the expression of >60% ofthe 308 genes differentially expressed between normal keratinocytes andmetastatic SCCs. NF-�B directly or indirectly modulated expression ofprograms of genes functionally linked to proliferation, apoptosis, adhe-sion, and angiogenesis. Among these, changes in expression of cyclin D1,inhibitor of apoptosis-1, mutant Trp53, and �-catenin detected with mod-ulation of NF-�B by microarray were confirmed by Western and North-ern blot. NF-�B DNA binding motifs were detected in the promoter of�63% of genes showing increased expression and 33% of the genesshowing decreased expression. The ACTACAG motif implicated in theNF-�B-dependent down-regulation of mRNA expression of MyoD andSox9 was detected in the coding portion of about 15% of genes showingincreased or decreased expression. Inactivation of NF-�B inhibited ma-lignant phenotypic features including proliferation, cell survival, migra-tion, angiogenesis, and tumorigenesis. These results provide evidence thatNF-�B is an important modulator of gene expression programs thatcontribute to the malignant phenotype of SCC.

INTRODUCTION

Many cancers share a complex set of phenotypic traits, suggestingthat common molecular regulatory mechanisms underlie their devel-opment (1). These traits include inhibition of programmed cell deathand increased proliferation, migration, new blood vessel formation(angiogenesis), and inflammation. The resemblance of these complexphenotypic changes in cancer to those occurring transiently during theresponse to injury and infection suggests the hypothesis that onco-genic activation of injury response pathways and their target genesmay be important in the pathogenesis of cancer. Consistent with thishypothesis, our investigations of molecular alterations in squamouscell carcinomas (SCCs) that arise from the skin and upper aerodiges-tive tract have revealed constitutive expression of multiple integrincell adhesion molecules and proinflammatory cytokines that are con-ditionally expressed in response to injury (2, 3). A wider molecularcomparison between normal, transformed, and metastatic keratino-cytes by mRNA differential display and cDNA microarray revealedaltered expression of an even greater diversity of genes, includinggenes functionally related to growth, apoptosis, adhesion, angiogen-esis, and inflammation (4). We noted that a number of these cytokines,cell adhesion molecules, and other genes expressed in SCC have been

associated with activation or as targets of the early injury responsetranscription factor, nuclear factor (NF)-�B (4–7).

NF-�B is expressed in an inactive form in most cells, composed ofNF-�B p50 and Rel A p65 subunits, and bound to an inhibitoryprotein, I�B� (8). In response to DNA damage, cytokine, or growthfactor signaling, I�B� is phosphorylated, ubiquitinated, and degradedby the proteasome, enabling nuclear localization and binding of p50/p65 to the promoter region of target genes. We established thatp50/p65 is the predominant form of NF-�B constitutively activated inhuman head and neck and murine SCCs and that NF-�B promotes theoverexpression of the proinflammatory and angiogenesis factor ho-mologues interleukin-8 and growth regulated oncogene-1 [Gro-1 (5,6)]. Inhibition of NF-�B by a dominant negative I�B� phosphoryla-tion mutant (I�B�M) or pharmacological antagonists inhibited tumorcell survival, cytokine expression, angiogenesis, and tumorigenesis byhuman and murine SCC (5, 7, 9, 10).

Molecular profiling of changes in gene expression with stepwisetransformation and metastatic progression of SCC in the syngeneicmurine Pam 212 model revealed altered expression of a diversity ofgenes, including a cluster of overexpressed genes in lymph nodemetastases (Pam LY cells) related to the NF-�B pathway (4). How-ever, we observed that the inhibitory effects on cell survival ofexpression of I�B�M under a constitutive promoter made it difficultto obtain stable transfectants of Pam LY and other SCCs for the studyof the role of NF-�B in gene expression and the malignant phenotype(7).

Here, we examined the overall differences in gene expressionbetween normal, transformed, and metastatic keratinocytes using15,000 cDNA microarrays, and we explored the role of NF-�B in thegenetic and phenotypic changes acquired during tumor progression inthe metastatic cells by genetically switching off NF-�B with I�B��under control of a doxycycline [DOX (Tet)]-inducible promoter. Weobtained evidence that NF-�B modulates a broad program of genesdifferentially expressed with tumor progression of this SCC. Wefurther established that inhibition of NF-�B inhibited proliferation,cell survival, migration, angiogenesis, and tumorigenesis of SCCcells. These results provide evidence that NF-�B is an essentialmolecular switch for the gene expression program and malignantphenotype in SCC.

MATERIALS AND METHODS

Cell Lines and Culture Conditions. The murine SCC line Pam 212 wasderived from spontaneously transformed BALB/c keratinocytes, and LY-2 wasisolated from lymph node metastases that developed in BALB/c mice afterinoculation of the parental PAM 212 SCC line (11). LY-2 has been shown togrow more aggressively and have a higher constitutive NF-�B activity than thePAM 212 line (5, 12). The SCC lines were maintained in Eagle’s MinimalEssential Medium (EMEM) (Life Technologies, Inc., Grand Island, NY) with10% fetal calf serum, 1% glutamine, and 0.5% penicillin/streptomycin at 37°Cin 5% CO2. Cell lines were determined to be Mycoplasma-free by nestedreverse transcription-polymerase chain reaction.

Transfection of Tet-On and Tet-Inducible I�B�M Plasmids. TheTet-On expression system used was obtained from Clontech Laboratories, Inc.,

Received 3/9/04; revised 5/26/04; accepted 7/15/04.Grant support: C. Van Waes was supported by NIDCD Intramural Project DC-00016.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Carter Van Waes, Head and Neck Surgery Branch, NationalInstitute on Deafness and Other Communication Disorders, Building 10, Room 5D55, 10Center Drive, MSC-1419, Rockville, MD 20892-0001. E-mail: [email protected].

©2004 American Association for Cancer Research.

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(Palo Alto, CA). LY-2 cells were cotransfected with the Tet-On plasmidsystem containing the Tet-responsive transcriptional activator that binds theTet-responsive element (TRE) and the gentamicin resistance gene (neo), to-gether with the tetR plasmid that binds the TRE in the absence of DOX thatreduces background levels of transcription. Cells (1.25 � 106) were plated into100-mm dishes, incubated overnight, and transfected with 1.6 �g of the Tet-Onplasmid, 14.4 �g of tetR, and 128 �L per reaction of LipofectAMINE (LifeTechnologies, Inc., Gaithersburg, MD) in Opti-MEM (Life Technologies, Inc.,Grand Island, NY). Clones containing the Tet-On plasmid were selected bylimiting dilution in 96-well plates using selection medium containing 600�g/mL G418. Clones were expanded and screened for responsiveness to DOXusing a TRE-luciferase assay. Fourteen clones with low background and highfold induction of luciferase reporter activity after the addition of 2 �g/mLDOX were selected and pooled as controls (LY-2P) and for transfection by theresponse plasmid containing I�B�M. The 14 LY-2 clones containing theTet-On plasmid were transferred into 6-well plates at a concentration of2 � 105 cells/well and incubated overnight at 37°C. The cells were transfectedwith 1 �g of TRE-I�B�M plasmid (pTRE-Igs32s36 from Clontech Labora-tories, Inc.), 4 �L of LipofectAMINE, and 6 �L of PLUS reagent (LifeTechnologies, Inc., Gaithersburg, MD) per reaction in Opti-MEM. Reactionswere incubated for 5 hours at 37°C in 5% CO2. The medium was changed tocomplete EMEM (Life Technologies, Inc.), and cells were allowed to recoverovernight. To select subclones containing both the Tet-On and TRE-I�B�Mplasmids, transfectants were diluted 1:40 for subcloning using one 200-mmdish for each clone. After 7 days in EMEM containing 600 �g/mL G418,colonies were selected using cloning cylinders (Genechoice, Frederick, MD),and four subclones from each dish, labeled A to D, were transferred to a24-well plate and expanded for screening for Tet-inducible expression ofI�B�M by Western blot, as described previously (5).

Nuclear Factor-�B Luciferase Assay. Four clones selected for expressionof I�B�� by Western blot and the LY-2P parent cells were transferred totriplicate wells at a concentration of 2 � 105 cells/well in 6-well tissue cultureplates. Cells were incubated overnight at 37°C and then transfected with 0.95�g of NF-�B luciferase plasmid, 4 �L of LipofectAMINE, and 6 �L of PLUSreagent in 1 mL of Opti-MEM per well for 5 hours at 37°C. The transfectionmedium was replaced with complete EMEM, and cells were incubated untilthey reached 80% confluence. NF-�B-luciferase assays were performed asdescribed previously (10).

Electromobility Shift Assay. Double-stranded DNA probes for NF-�B(5�-AGTTGAGGGGACTTTC-CCAGGC-3�) and Oct-1 (5�-TGTCGAATG-CAAATCACTAGAA-3�) were commercially obtained (Promega, Madison,WI). Probes were labeled with [�-32P]ATP (6000 Ci/mmol; Amersham, Ar-lington Heights, IL). Nuclear extracts were prepared from DOX-treated anduntreated LY-2 I�B�� clones, and DNA binding assays were performed with5 �g of nuclear extracts using standard methods described previously (5).

Microarray Analysis. Total RNA was isolated from primary BALB/ckeratinocytes, Pam 212, and LY-2 cells and the LY-2P and the four inducibleLY-2 I�B�� clones (23D, 24C, 25D, and 26C) with and without DOXtreatment. RNA was collected 72 hours after DOX treatment in three inde-pendent experiments. The 15,000-element murine microarray developed by theRadiation Oncology Sciences Program, National Cancer Institute, using acDNA library from the National Institute of Aging (Mm-ROSP-NIA15K), wasused.4 Sequential arrays were obtained from two different printings. Experi-ments were performed in triplicate. The protocols used for RNA isolation,hybridization, and microarray analysis were those available from the NationalHuman Genomics Research Institute.5 Arrays were read on a Genepix 4000BScanner, and data were collected using Genepix Pro 4.0 software (AxonInstruments, Foster City, CA).

Statistical and Bioinformatic Analyses. Array elements exhibiting meansignal differences of �2-fold (99% confidence interval) for triplicate compar-isons among cDNAs from normal keratinocytes, transformed Pam 212, and/ormetastatic Pam LY-2 cells were scored as being associated with tumor pro-gression. Array elements exhibiting reversion of mean differences of �2-fold(99% confidence interval) for comparisons among cDNAs from metastaticPam LY-2P and four LY-2 I�B�M clones � DOX were scored as putativeNF-�B-modulated genes. Analysis of the collected data was performed using

the Cluster and Treeview software developed by Michael Eisen (StanfordUniversity), as described previously (4). Genes were assigned to functionalfamilies based on information from LocusLink and PubMed. Sequence datagenerated through the use of the Celera Discovery System and verified inpublic databases were used for detection of NF-�B–related motifs. The se-quences from 2 kb 5� and the full-length of each gene found to be differentiallyexpressed by microarray were examined for NF-�B binding motifs (12) byGenomatix software and for ACTACAG motifs involved in NF-�B–mediateddown-regulation of MyoD and Sox9 mRNA (13) by BLAST search availablefrom the National Center for Bioinformatics, National Institutes of Health.

Northern Blot. RNA isolated for microarray analysis was subjected toelectrophoresis and Northern blot as described previously (4). Blots wereprobed with full-length 32P-labeled DNA probes for the cyclin D1, Trp53,inhibitor of apoptosis-1 (IAP-1), �-catenin, and �-actin genes and washedunder high stringency conditions.

Whole Cell Lysates and Western Blot. Whole cell lysates were collectedfrom DOX-treated and untreated I�B�� clones in 6-well plates after washingtwice with PBS and addition of 300 �L of lysis buffer [100 mmol/L potassiumphosphate, 0.2% Triton X-100, and 0.05% dithiothreitol (Tropix; PE Biosys-tems, Bedford, MA)] to each well. Plates were held at 4°C for 1 hour and thensubjected to three freeze-thaw cycles. Lysates were transferred to 2-mL mi-crotubes and centrifuged for 5 minutes at 13,000 rpm. Lysates were diluted1:10 in distilled H2O and analyzed for protein content by the BCA proteinassay kit (Pierce, Rockford, IL). Samples were stored at �80°C until use.Twenty micrograms of protein were loaded onto precast 12% Tris-glycine gels(Novex, San Diego, CA). Gels were run for 2 hours at 100 V, and then proteinswere transferred to nitrocellulose membranes for 90 minutes at 20 V. Mem-branes were blocked in 5% milk in Tris-buffered saline at 4°C overnight. Forselection of clones expressing I�B�� after DOX treatment, blots were incu-bated for 2 hours at 4°C in a 1:200 dilution of anti-I�B� (rabbit polyclonalantibody; Santa Cruz Biotechnology, Santa Cruz, CA). Antibodies recognizingcyclin D1, Trp53, IAP-1, �-catenin, and �-tubulin proteins were obtained fromSanta Cruz Biotechnology. Membranes were washed and incubated for 1 hourat room temperature in a 1:3,000 dilution of goat antirabbit antibody labeledwith horseradish peroxidase (Santa Cruz Biotechnology). Membranes werewashed, enhanced with SuperSignal chemiluminescence reagent (Pierce),placed in heat-sealable pouches (Kapak, Minneapolis, MN), and exposed tofilm.

MTT Cell Proliferation Assay. Proliferation of LY-P and the I�B��clones was measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide (MTT) cell proliferation kit (Roche Molecular Biochemicals,Mannheim, Germany). Cells were transferred in triplicate to a 96-well, flat-bottomed plate at a concentration of 5 � 103 cells/well in 100 �L of completeEMEM with or without DOX. Cells were incubated at 37°C in 5% CO2 for 1,3, or 5 days, and growth rates were analyzed after the addition of MTT reagentto the cultured cells and lysis according to the manufacturer’s instructions.Absorbance was determined using multichannel spectrophotometry at a wave-length of 570 nm.

Cell Cycle Analysis. LY-2P, 23D, and 24C cells were transferred to 6-wellplates at a concentration of 5 � 105 cells/well and treated with 2 �g/mL DOXor left untreated. After 96 hours, floating and adherent cells were collected andcounted. One million cells were washed twice in buffer solution from theCycleTest Plus DNA Reagent Kit (Becton Dickinson, San Jose, CA) and thenresuspended in 250 �L of trypsin buffer. Cells were incubated for 10 minutesat room temperature. Two hundred microliters of trypsin inhibitor/RNasebuffer were added to each tube, and cells were incubated for 10 minutes atroom temperature. Two hundred microliters of cold propidium iodide stainingsolution were added. Cells were incubated for 10 minutes in the dark on ice,and within 3 hours of staining, quantification of DNA was determined on aFACScan flow cytometer using CellQuest software (Becton Dickinson).

Invasion Assays. Invasion assays were performed using a synthetic base-ment membrane cell invasion assay kit (Chemicon, Temecula, CA). Cells wereplated at a concentration of 0.5 � 106 cells/mL in serum-free EMEM into theinserts of a cell invasion assay plate. Plates were incubated at 37°C in 5% CO2

for 72 hours. Cells on the interior of the inserts were removed by swabbing,and the exterior of the inserts was stained for evidence of cell migrationthrough the synthetic basement membrane. Stained cells were solubilized in10% acetic acid, and the intensity of staining was quantified by transfer of

4 http://nciarray.nci.nih.gov.5 http://research.nhgri.nih.gov/microarray/Protocols.pdf.

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NF-�B IN SQUAMOUS CELL CARCINOMA



100-�L aliquots to a 96-well plate for spectrophotometry at a wavelength of570 nm.

Determination of Tumor Growth and Microvessel Density in SevereCombined Immunodeficient Mice. LY-2P cells (5 � 106) as a control andLY-2 24C cells were inoculated in 200 �L of serum-free EMEM subcutane-ously into the flanks of congenic male BALB/c severe combined immunod-eficient (SCID) mice (5 mice per group). Animals received water treated with2 �g/mL DOX beginning at week 7. Tumor measurements were recorded oncea week for the duration of tumor growth to 10 weeks. After euthanasia, tumorswere harvested, prepared for frozen section, and stained with hematoxylin andeosin (H&E) or antibody specific for CD31 as described previously (9).

RESULTS

Inhibition of Nuclear Factor-�B in Squamous Cell Carcinomaby Tet-Inducible I�B�M. A stepwise model of cancer developmentand progression was established previously from syngeneic BALB/cmice consisting of normal keratinocytes (KER), a transformed SCCcell line (Pam 212), and SCC lymph node metastases, designated PamLY (4, 11). Pam 212 cells produce slowly growing SCC that rarelymetastasizes, and Pam LY cells isolated from rare lymph node me-tastases of Pam 212 are variants that form cancers that grow andmetastasize rapidly. Molecular comparisons between these normal,transformed, and metastatic keratinocytes by mRNA differential dis-play and a first-generation 4,000-element cDNA microarray providedevidence for altered expression of multiple genes related to growth,apoptosis, angiogenesis, inflammation, and the NF-�B pathway (4).

To directly explore the role of NF-�B in the accumulated changesin gene expression acquired with tumor progression of Pam LY, wesequentially transfected plasmids containing a TRE and the I�B signalmutant I�B�M under the control of a DOX-inducible promoter intothe Pam LY-2 line, a representative metastatic line that exhibitsconstitutive activation of NF-�B (5). Multiple LY-2 cell clones thatshowed low background and high inducibility by the Tet derivativeDOX in a Tet reporter assay after first-stage transfection were pooledfor transfection by the Tet-I�B�M construct and for use as controls.

Expression of I�B�M by LY-2 clones was detected by Western blotas described previously (5), and four clones that exhibited low basaland strong DOX-induced expression of I�B�M were selected toexamine effects on NF-�B activation. The effect of DOX-inducedI�B�M expression on constitutive NF-�B-luciferase reporter geneactivity in the four LY-2 subclones and LY-2P control lacking Tet-I�B�M is shown in Fig. 1A. I�B�M inhibited NF-�B functionalpromoter activity as determined using NF-�B-luciferase reporter as-say (Fig. 1A). Progressive inhibition of constitutive NF-�B reporteractivity to �95% of baseline was observed by day 4 after addition ofDOX to LY-2 I�B�M clones. NF-�B reporter activity in controlLY-2P cells lacking Tet-I�B�M was not suppressed below controllevels (Fig. 1A). I�B�M also suppressed NF-�B DNA binding activ-ity in nuclear extracts of LY-2 clones in electromobility shift assay(Fig. 1B). The specificity and composition of constitutive NF-�Bp50/Rel Ap65 DNA binding activity in LY-2 cells were reported previ-ously (5).

Molecular Profile of Genes Related to Tumor Progression andNuclear Factor-�B in Squamous Cell Carcinoma. To directly ex-plore the alterations and role of NF-�B in gene expression with tumorprogression, we used the National Cancer Institute 15,000-element cDNAmicroarray expressing known genes to examine the diversity of genesdifferentially expressed among keratinocytes, transformed Pam 212, andmetastatic Pam LY-2 (Met) SCC cells, and we determined the effect ofconditionally inhibiting NF-�B in Pam LY-2 cells by expressing I�B�M.Complementary DNA was prepared from mRNA from three independentpreparations of keratinocytes, Pam 212, and Pam LY-2 cells and fromfour Pam LY-2 clones expressing I�B�M after culture with DOX for 72hours. Messenger RNA was isolated at 72 hours based on the �75%inhibition of NF-�B reporter activity detected in Fig. 1 and to minimizecell death or RNA degradation, which was observed beyond 96 hours(Figs. 3 and 5; data not shown). Fig. 2 shows the cluster analysis for 308genes (horizontal rows) that exhibited �2-fold difference in expressionamong keratinocytes (Ker), transformed Pam 212 (Trans), or metastatic

Fig. 1. Inhibition of NF-�B in LY-2 SCC clones by Tet-inducible I�B�M. A, NF-�B-luciferase reporter assay. LY-2P pooled control clones and LY-2 Tet-I�B�M clones 23D, 24C,25D, and 26C were transfected with a NF-�B-luciferase reporter and incubated � DOX as described in Materials and Methods. NF-�B-luciferase reporter activity measured on days0, 2, and 4 is shown. B, NF-�B DNA binding assay. LY-2 Tet-I�B�M clones 23D, 24C, 25D, and 26C were incubated � DOX for 72 hours, and cell extracts were subjected toelectromobility shift assay with consensus NF-�B or control Oct-1–specific oligonucleotides as described. The specificity and composition of constitutive NF-�Bp50/Rel Ap65 DNAbinding activity in LY-2 cells were reported previously (5).

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Pam LY-2 (Met) cells and Pam LY-2 cells expressing I�B�M after DOX(vertical columns). Overall, the differential pattern of expression detectedfor 308 of 15,000 of the gene elements on the array represents about 2%of the genes compared. Three dominant patterns were detected thatclustered according to the stepwise differences in phenotype amongKER, Pam 212, and Pam LY-2 cells. Cluster 1 genes showed similarexpression in normal and transformed Pam 212 cells but reduced expres-sion in metastatic LY-2 cells. Cluster 2 genes showed lower expression innormal cells but increased expression in transformed and metastatic cells.Cluster 3 genes showed increased expression primarily in LY-2 meta-

static cells. Overall, 141 genes in the upper cluster showed a decrease,and 167 genes in clusters 2 and 3 were increased in association withtransformation, tumor progression, and activation of NF-�B.

Tables 1 and 2 show a selected list from the 308 genes detected bymicroarray and cluster analysis that exhibited �2-fold difference inhybridization with malignant progression and/or inhibition of NF-�B(the full list is available online).6 Table 1 includes the genes that show

6 http://www.nidcd.nih.gov/research/scientists/vanwaesc.asp.

Fig. 2. Cluster analysis of genes related to tumorprogression and NF-�B in SCC. ComplementaryDNA was prepared from mRNA from three inde-pendent preparations of keratinocytes, Pam 212,and Pam LY-2 cells and four Pam LY-2 clonesexpressing I�B�M after culture with DOX for 72hours. A total of 308 of 15,000 cDNA microarrayelements exhibited mean signal differences of �2-fold (99% confidence interval) for triplicate com-parisons between cDNAs from normal keratino-cytes (Ker), transformed Pam 212 (Trans), and/ormetastatic Pam LY-2 (Met) cells. Three dominantpatterns were detected that clustered according tothe stepwise differences in phenotype among KER,Pam 212, and Pam LY-2 cells. Cluster 1 genesshowed similar profiles in keratinocytes and trans-formed Pam 212 cells (green) but reduced expres-sion in metastatic LY-2 cells (red). Cluster 2 genesshowed lower expression in normal cells (red) butincreased expression in transformed and metastaticcells (green). Cluster 3 genes showed low intensityin keratinocytes and transformed cells (red) andincreased signal intensity primarily in LY-2 meta-static cells (green).

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Table 1 Selected list of genes increased with tumor progression

FunctionGene Symbol Clone ID NF-�B association

Fold change

LY-2/Ker LY-2/I�B-�M

Cell cycle/growthCyclin D1* Ccnd1 H3084D05 Target gene 3.351 �2.266Cyclin D2* Ccnd2 H3152D01 Target gene 2.957 �2.05Growth arrest specific 5* Gas5 H3113A12 6.304 �7.813Milk fat globule-EGF factor 8* Mfge8 H3126F11 Target gene 2.594 �3.525Protein phosphatase 3*† Ppp3cb H3065C08 Inhibitor of NF-�B 3.117 �2.231Proliferating cell nuclear antigen* Pcna H3021F12 Target gene 5.775 �2.811

ApoptosisBaculoviral IAP repeat* Birc2 H3074A02 Target gene 9.154 �6.013Bcl-2 related ovarian killer*† Bok1 H3081D02 1.994 �3.461Immediate early response 3* Ier3 H3057B07 Target gene 2.865 �3.851Transformation related protein*† Trp53 H3142D07 Target gene 2.9 �4.142Uchrp† Uchrp IMAGE:605056 2.188 �4.353

Inflammation/angiogenesisColony stimulating factor 1* Csf1 H3057D05 Target gene 14.639 �5.574Complement component 3*† C3 H3054A08 Target gene 5.145 �8.303FGF receptor* Fgfr4 IMAGE:406823 Target gene 1.982 �1.991Gro 1 oncogene* Gro1 H3051F10 Target gene 12.394 �4.094Histocompatibility 2-L* H2-L H3096A12 Target gene 2.342 �3.431Histocompatibility 2-D* H2-D H3141B11 Target gene 2.968 �3.927Interferon receptor* Ifnar H3118F09 Inhibitor of NF-�B 4.5 �3.054Lymphocyte antigen complex* Ly6e H3027D05 Inducer of NF-�B 4.029 �2.613

MetastasisIntegrin � 3*† Itga3 H3137A03 Inducer of NF-�B 15.597 �3.917Laminin � 5*† Lama5 H3002G01 Target gene 2.272 �2.967Laminin receptor 1 Lamr1 H3075G08 2.169 �1.619Plasminogen activator, tissue Plat H3080H11 Target gene 2.667 �2.487Procollagen type 5 � 2 Col5a2 H3156E09 Target gene 5.103 �2.094Syndecan 1* Sdc1 H3013F05 Target gene 2.948 �2.034

MetabolismATPase H� transport*† Atp6b H3120H04 Inhibitor of NF-�B 2.247 �2.858Branched chain ketoacid dehyd* Bckdk H3136B09 2.455 �3.042Choline kinase*† Chk H3088E07 Inducer of NF-�B 6.662 �3.252Cytochrome p450* Cyp1b1 J0216F07 Target gene 22.289 �4.286Glutathione-S-transferase* Gstm1 H3133A06 Target gene 3.037 �3.061Low density lipoprotein receptor* Ldlr H3014C04 Target gene 2.689 �3.226Mannose-6-phosphate receptor† M6pr H3092C05 Inhibitor of NF-�B 6.303 �3.612Potassium intermediate* Kcnn4 H3054H04 2.778 �2.197Solute carrier family 12*† Slc12a2 H3077B02 2.551 �2.145

Stress responseHeat shock protein, 70 kDa* Hspa5 H3032A08 Activates NF-�B 14.303 �6.369Heat shock protein 84 kDa* Hsp84 H3042G07 Activates NF-�B 3.437 �7.198Heat shock protein 86 kDa* Hsp86 H3023G01 Activates NF-�B 2.61 �2.34Heat shock protein cognate 70* Hsc70 H3133H01 Binds NF-�B 3.42 �10.229Superoxide dismutase* Sod1 H3130B11 Target gene 4.784 �2.145

Signal transductionAXL receptortyrosine kinase*† Axl H3152F05 Inhibitor of NF-�B 2.459 0.938CD97 (EGF-TM7)*† Cd97 H3032G06 2.283 �1.352Interleukin-1 receptor associated* Il1rak H3042E08 Activates NF-�B 1.999 �1.296Frizzled 7 homolog Fzd7 H3031A03 2.717 �0.843Growth arrest & DNA damage specific* Gadd45g H3054C02 Activates NF-�B 3.144 �1.407Growth factor receptor bound* Grb2 H3153D02 Activates NF-�B 2.483 �2.341N-myc downstream regulated* Ndr2 G0110H06 Inhibits p50 2.248 �2.039PI3 kinase regulatory* Pik3r1 H3067B08 Activates NF-�B 4.206 �3.539Protein tyrosine phosphatase*† Ptpn13 H3118G02 Inhibitor of NF-�B 2.844 �9.071Ras p21 protein activator 3*† Rasa3 H3054E01 Activates NF-�B 3.223 �2.71Ras-related C3* Rac1 IMAGE:477981 2.042 �3.875Transferrin receptor* Trfr H3059G03 2.216 �1.145

Nuclear proteins/transcription factorsActivating transcription factor† Atf2 J0221F08 1.606 �2.696Breast cancer, early onset† Brca2 H3069F08 2.216 �0.604Butyrate response factor* Brf2 H3015E08 2.072 �2.008High mobility group AT* Hmga1 H3029B11 Target gene 2.816 �2.591Jerky* Jrk H3119F06 2.849 �3.329Myelocytomatosis oncogene* Myc H3089H11 Target gene 2.224 �2.42Nuclear factor �B p105* Nfkb1 H3072E09 2.919 �1.443Sex comb on midleg-like 1† Scml1 H3113B01 2.355 �1.154Yes-associated protein 65 kDa Yap H3089H07 2.072 �1.746

RNA processingDEAD box protein 3* Ddx3 H3018F11 2.976 �2.055DJ-1 protein† DJ-1 H3150D06 3.155 �4.611FGF inducible 14 Fin14 H3018G01 2.358 �3.355Nuclear ribonuclease Hnrpa1 H3111H11 4.926 �2.785RNA polymerase 1-1* Rpo1-1 H3049D09 2.703 �2.328

Protein synthesis/modificationERO1 like*† Ero1l H3126B01 2.529 �2.456Ribosomal protein L27a Rpl27a H3009B05 2.183 1.369Ribosomal protein L8 Rpl8 H3141F09 2.778 1.005Ribosomal protein S18* Rps18 H3006C11 2.487 �2.424Ubiquitin activating enzyme E1 Ube1x H3022E03 Phosphorylates I�B 5.313 �3.139

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increased hybridization, and Table 2 includes the genes that showdecreased hybridization by cDNA of Pam 212 and/or LY-2 cells. Thegenes were classified according to putative function and publishedassociations with NF-�B as determined by search of National Centerfor Biotechnology Information PubMed and LocusLink. The ratio ofLY-2 to keratinocyte hybridization, representing the overall changewith malignant progression, and the ratio of LY-2 to LY-2 I�B�M,representing the effects of expression of I�B�M, are shown. Thegenes that are modulated by �2-fold by I�B�M are shown in bold.

Remarkably, Fig. 2 and Tables 1 and 2 reveal that expression ofI�B�M in Pam LY-2 results in restoration of the pattern of expressionobserved for keratinocytes for many of the genes up- and down-regulated between keratinocytes and Pam LY-2 metastatic cells. Ofthe genes up-regulated in LY-2 cells, hybrization of 115 of 167 (69%)decreased by �2-fold toward normal levels after expression ofI�B�M. Fully 88 of 141 (62%) genes down-regulated in LY-2 cellswere restored by �2-fold back toward the level observed in kerati-nocytes after expression of I�B�M. The effect of I�B�M to up-modulate as well as down-modulate mRNAs provides evidence thatthe wide effect on gene expression is not merely due to degradation ofmRNA during the process of cell death, and we verified the viabilityof cells and integrity of mRNA after 72 hours of exposure to DOX(data not shown).

Corroboration between the Gene Profile Expressed in LY-2Cells and Previously Identified Squamous Cell Carcinoma-Related Genes. As an initial assessment of the consistency andvalidity of the differential gene expression profile obtained for LY-2-expressing cells using the 15,000-element cDNA microarray, weidentified genes that were previously found to be differentially ex-pressed in multiple LY metastasis cell lines by a first-generation4,000-element murine cDNA microarray and confirmed by reversetranscription-polymerase chain reaction and/or Northern blot analysis(4). Several genes in Table 1 that were classified as increased in LY-2cells by �2-fold were included and detected in both array studies.These include the increase in expression of proliferation and antiapo-

ptosis-related genes cyclin D1 (Ccnd1), proliferating cell nuclearantigen (Pcna), inhibitor of apoptosis-1 (IAP-1/Birc-2), and growtharrest-specific-5 (Gas5); inflammatory factors and receptors growth-regulated oncogene 1 (Gro-1/KC), colony-stimulating factor-1 (csf1),complement 3 (C3), and lymphocyte antigen 6e (Ly6e); metabolismand drug resistance genes cytochrome P450 (Cyp1b1) and glutathioneS-transferase (Gstm1); signal molecules protein tyrosine phosphatase(Ptpn13) and Yes-associated protein 65 kDa (Yap65); and chromatin-associated high mobility group protein [Hmga1 (4)]. Of these, differ-ential expression in LY-2 cells and two other LY lines was alsoconfirmed previously by Northern blot for Gas5, Gro-1/KC, Ly6,Gstm1, and Yap65 (4, 14). Several genes in Table 2 that wereclassified as decreased in LY-2 cells by �2-fold were also detectedusing both arrays. These include cyclin-dependent kinases 1 and 4(Cdkn1 and Cdk4), calmodulin (Calm), cadherin 1 (Cdh 1), prothy-mosin b4 (Ptmb4), mucin (muc18), procollagens type I and IV�(Col1a2 and Col4a2); ATPase (Atp11a), and creatine kinase (Ckmt1).Decreased expression of Ptmb4 was confirmed previously by North-ern blot (4). Two elements showed discordant expression between thetwo studies, tropomyosin � (Tpm1) and A disintegrin/MMP (Adamts-1). Overall, a consistent pattern of expression was observed for themajority of elements found to be differentially expressed by cDNAhybridization in these independent studies and for six of eight genesconfirmed by Northern blot (4).

Effect of I�B�M on Expression of Cyclin D1, Trp53, Inhibitorof Apoptosis-1, and �-Catenin. Changes in expression of several ofthe genes classified by microarray as differentially expressed in LY2cells and modulated in LY2 I�B�M-expressing cells have been de-tected with oncogenesis and metastatic progression of SCC. CyclinD1, IAP-1, and mutant Trp53 have been reported to be overexpressedin advanced SCC, and �-catenin expression has been reported to bedecreased in advanced SCC, consistent with the expression profile forthese genes in LY-2 cells (Tables 1 and 2). Cyclin D1, Trp53, andIAP-1 may be up-regulated with NF-�B in other cancer lines, and�-catenin may be down-regulated with NF-�B in other cancer lines

Table 1 Continued


Fold change

LY-2/Ker LY-2/I�B-�M

Ubiquitin B Ubb H3138A08 Labels I�B 8.144 �1.171Ubiquitin conjugating enzyme E2 Ube2h H3057B09 Ubiquitinates I�B 3.333 �1.199Ubiquitin conjugating enzyme E3† Ube3a H3102B01 Ubiquitinates I�B 4.878 �5.096Ubiquitin specific protease 9† Usp9x H3139F12 3.769 �3.175

Structural proteinsAlpha tropomyosin* Tpm1 H3120G06 Binds p65 2.421 �4.36Cadherin* Cdh3 H3018F05 Inflammatory 2.328 �2.706Capping protein alpha 2 Cappa2 H3085F12 14.293 �7.458Dystroglycan 1* Dag1 H3008B05 Activates NF-�B 2.593 �4.738Epithelial protein lost*† Eplin H3153C05 Activates NF-�B 2.066 �2.971Fascin homolog 1* Fscn1 H3006D08 2.315 �2.581Four and a half LIM domains* Fhl2 H3033C07 2.169 �1.189Keratin complex 1 acidic* Krt1-18 H3021B02 Target gene 3.105 �5.517Keratin complex 2 basic* Krt2-8 H3031C01 2.094 �5.568PDZ and LIM domain 1* Pdlim1 J0824B03 5.7 �3.775Protocadherin 7*† Pcdh7 H3067F12 Inflammatory 6.556 �1.026Thymopoietin* Tmpo H3096B08 9.939 �8.153

OtherGlobin inducing factor† Gbif H3053F12 2.003 1.002Metallothionein 2 Mt2 H3013D11 Inhibits I�B degradation 2.145 �3.029Next to the Brca1*† Nbr1 H3061D04 2.367 �1.888RAN binding protein* Ranbp9 H3013A10 Accumulates I�B� 2.87 �3.189Repeat family 3 gene* Llrep3 H3107F07 3.556 �7.704Ring finger protein 19* Rnf19 H3153A08 Activates NF-�B 2.148 �4.402Sema domain, immunoglobin*† Sema3f H3134D09 2.191 �1.667Suppressor of Lec15† Supl15h H3090D12 2.001 �1.383TGF beta inducible transcript* Tgfb1i1 H3122H01 3.068 �2.974

NOTE. Total number of genes regulated by NF-�B 105/167. Total number of genes previously associated with NF-�B 67/167.� Genes containing �B site in promoter region.† Genes containing ACTACAG motif in coding sequence.

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Table 2 Selected list of genes decreased with tumor progression


Fold change

LY2/Ker LY2/I�B-�M

Cell cycle/growthC-src tyrosine kinase* Csk L0237H04 �2.693 2.358Calmodulin Calm H3006H05 Activates NF-�B via IKK �2.554 2.42Cell division cycle homolog 25a Cdc25a H3050E04 �2.341 2.269Cell division cycle homolog 45 Cdc45l H3003E07 �2.032 2.029Cyclin C Ccnc C0117F09 �2.739 2.418Cyclin E2* Ccne2 C0186A01 �2.309 2.155Cyclin dependent kinase 4 Cdk4 H3147D06 Target gene of NF-�B �2.734 2.05Cyclin dependent kinase inhibitor* Cdkn1c H3097D03 �5.208 2.913Platelet derived growth factor* Pdgfa H3146C02 �2.15 2.906

ApoptosisATP binding cassette* Abcd3 H3143E03 NF-�B site in promoter �2.89 2.535Bcl2/adenovirus E1B Bnip3 H3103B07 Transient inhibitor of NF-�B �2.364 2.436Fas associating w/ death domain Fadd H3095D08 Inducer of NF-�B �3.597 2.975

Inflammation/angiogenesisCoagulation factor III*† F3 H3014G02 Activates NF-�B via IKK �4.672 3.858Interleukin 17 receptor Il17r H3008A03 Activates NF-�B via MAPK �3.021 3.048Interleukin 2 receptor Il2ra J0052C08 �2.262 2.207Lymphocyte antigen 6 complex Ly6 H3115A08 Inducer of NF-�B �5.617 3.311Prothymosin � Ptmb4 H3143A02 �21.739 8.858

MetastasisA disintegrin/MMP Adamts1 H3034B07 �2.695 2.736Cadherin 1* Cdh1 H3076B06 Associated with inflammation �2.597 2.313Kangai 1† Kai1 H3154D02 Target gene of NF-�B �2.816 2.139Lipocalin 2 Lcn2 H3083G02 Associated with inflammation �2.506 11.235Procollagen type 1 � Col1a2 H3125D01 �4.901 2.886Procollagen type II � Col2a1 H3026G09 �6.896 2.139Procollagen type III � Col3a1 H3005D11 Inducer of NF-�B �3.205 4.268Secreted acidic C-rich Sparc H3026D08 �8.928 2.872Tissue inhibitor of MMPs Timp3 H3031E01 �3.3 3.289

MetabolismATPase, type 11A† Atp11a H3097B05 �2.888 2.734ATP synthase H� transport Atp5j2 H3118C01 Inhibitor of NF-�B upregulates I�B� normal half-life �2.191 2.111Glutathione peroxidase* Gpx3 J0088G08 �2.604 4.065Lipopolysaccharide binding*† Lbp H3086G08 Activates NF-�B via MAPK �2.977 2.103Phosphoprotein enriched† Pea15 H3014G07 Inducer of NF-�B �2.424 1.159Pyruvate dehydrogenase*† Pdha1 H3068G07 NF-�B site in promoter �3.021 1.643Sterol carrier protein 2*† Scp2 H3122F12 �2.412 1.488

Stress responseCrystallin � 2 Crya2 H3143B04 Inhibitor of NF-�B �3.921 3.479

Signal transductionAdenylate kinase† Ak2 H3052D11 �3.755 3.546Max dimerization protein 4*† Mad4 H3131B07 �2.008 2.131MAD homolog 4 Madh4 H3128C04 �2.765 2.114NF-�B enhancer inhibitor* Nfkbia H3026A08 �1.433 4.611NIK-related kinase Nrk H3008B02 Activates NF-�B �2.244 4.859Phosphoglycerate kinase* Pgk1 H3023D06 �2.659 2.061Protein tyrosine phosphatase4 Ptp4a2 H3088F03 �2.118 1.615Rho-associated coiled-coil Rock1 H3069C09 �2.808 3.183TNF receptor associated factor Traf1 H3015E06 NF-�B dependent �2.011 1.243

Nuclear proteins/transcription factorsCbp/p300 interacting transactivation Cited4 H3076H08 NF-�B co-activator �2.178 2.745High mobility group box 1 Hmgb1 H3126A05 Binds p50 subunit �3.104 1.739Jun oncogene Jun H3058C09 NF-�B co-activator �2.906 2.057Ras-related C3 Rac1 H3018C09 Inducer of NF-�B �2.004 3.329

RNA processingNuclear protein 220† Np220 H3029A07 �2.259 1.827RNA polymerase II* Rpo2-3 H3055H08 Co-activator of p65 �2.639 4.501

Protein synthesis/modificationEukaryotic translation 4g2 Eif4g2 H3113E10 �4.698 2.222Nedd4 WW-binding protein 4* N4wbp4 H3062G06 �6.966 2.814

Structural proteins� 2 glycoprotein 1*† Azgp1 IMAGE:521249 �2.013 1.398� spectrin 2*† Spnb2 H3010G09 �3.781 1.015Catenin �* Catnb H3031E05 Regulated by IKK �3.998 3.061Fibronectin† Fn1 H3116A10 �6.201 4.723Catenin � 1*† Catna1 H3018E08 �1.996 1.723Tenascin C Tnc L0062E01 �2.557 1.956

OtherDeleted in polyposis Dp1 J0420H06 �2.473 2.137Insulin-like growth factor r2 Igfr2 H3148G08 �2.765 2.494Ninjurin 1* Ninj1 H3072B10 �2.579 4.878Rabaptin 5*† Rab5ep H3002C01 �2.739 1.333Topoisomerase II �*† Top2a H3139A05 Inducer of NF-�B �2.427 2.893Tumor differentially expressed† Tde11 H3014H10 �2.593 1.18WW domain binding 5* Wbp5 H3127H02 �2.087 3.313Zinc finger protein 68*† Zfp68 H3058F07 �4.122 5.885

NOTE. Number of genes regulated by NF-�B 47/141. Number of genes previously associated with NF-�B 39/141.� Genes containing �B site in promoter region.† Genes containing ACTACAG motif in coding sequence.

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(15–19), consistent with increased activation of NF-�B (5) in LY-2SCC cells (Tables 1 and 2).

To validate the expression profile of cyclin D1, Trp53, IAP-1, and�-catenin detected in LY-2 cells and that obtained after modulation ofNF-�B by I�B�M, expression was compared by Western and North-ern blot analysis using protein and mRNA extracts from samplesobtained after 72 hours of culture � DOX, concurrent with arrayexperiments shown in Fig. 1. Fig. 3A shows Western blots comparingcyclin D1, Trp53, IAP-1, and �-catenin protein expression in the lowNF-�B Pam 212, high NF-�B LY-2 cells and in three LY-2 I�B�Mclones � DOX. Compared with Pam 212, LY-2 cells and most of theLY-2 I�B�M clones without DOX showed increased cyclin D1,IAP-1, and Trp53 and decreased �-catenin protein expression levels,consistent with the profiles obtained by array. Conversely, with inhi-bition of NF-�B by I�B�M in the LY-2 clones � DOX, decreasedexpression of cyclin D1, Trp53, and IAP-1 and increased expressionof �-catenin proteins are observed in most clones, consistent withmodulation of the genes encoding these proteins. There were nodifferences due to differences in loading or NF-�B activation betweencell lines when blots were probed for the constitutively expressedprotein �-tubulin. Thus, the Western blot data are consistent with the

role of NF-�B in the modulation of these genes in LY-2 cells detectedby microarray.

To establish whether cyclin D1, Trp53, IAP-1, and �-cateninmRNA expression was modulated with NF-�B in LY2 I�B�M-expressing cells, Northern blot analysis was performed to comparemRNA expression by the LY2 I�B�M clone 24C cultured � DOX.The Northern blots in Fig. 3B show that expression of I�B�M in LY-224C cells � DOX resulted in decreased mRNA signal for cyclin D1,Trp53, and IAP-1 compared with LY-2 24C cells without DOX. Incontrast, �-catenin mRNA expression was increased after expressionof I�B�M in LY-2 24C cells � DOX. No nonspecific induction ordegradation of mRNA or differences in loading were detected whenblots were probed with �-actin. The differences in protein and mRNAexpression for the LY2 I�B�M clone 24C cultured � DOX arequantified and compared by densitometry in Fig. 3C and D. Thedirection of change for the proteins and genes studied is consistent forall four genes. The difference in protein and mRNA expression wassimilar for cyclin D1 and �-catenin, whereas the percentage differencefor protein exceeds that observed for mRNA at the 72-hour timeinterval for IAP-1 and p53. Together, the Western and Northern blotdata validate the expression profile detected by array for these known

Fig. 3. Expression of cyclin D1, IAP-1, Trp53, and �-catenin with tumor progression and after suppression of NF-�B activation by I�B�M. A, Western blot analysis of proteinexpression in Pam 212 and LY-2 control and in LY-2 I�B�M clones 23D, 24C, and 25D � DOX. Increased protein expression of cyclin D1, IAP-1, and Trp53 and decreased expressionof �-catenin with increased activation of NF-�B were observed in LY-2 cells relative to Pam 212 cells. Cyclin D1, IAP-1, and Trp53 protein expression is inhibited and �-cateninexpression is increased in LY-2 I�B�M clones after DOX-induced expression of I�B�M. �-Tubulin served as a control for loading and integrity of protein. B, Northern blot analysisof mRNA expression in LY-2 24C Tet I�B�M � DOX. The mRNA expression of cyclin D1, Trp53, and IAP-1 is inhibited in LY-2 24C cells after DOX-induced expression of I�B�M.Levels of �-catenin mRNA are increased in LY-2 24C cells after DOX-induced expression of I�B�M. �-Actin serves as a control for loading and integrity of mRNA. C and D,densitometric comparison of protein and mRNA expression of cyclin D1, IAP-1, Trp53, and �-catenin. The percentage difference in protein and mRNA expression between �DOXand �DOX normalized to constitutive controls is shown. f, 24C�; �, 24C�.

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cancer-related genes in LY-2 cells and confirm that modulation ofNF-�B by I�B�M results in up- or down-modulation of severalNF-�B–regulated genes.

Because expression of wild-type Trp53 has been reported to haveproapoptotic rather than antiapoptotic effects, we performed a se-quence analysis of Trp53 in LY-2. This established the presence ofnonconserved mutations in exons 6, codon 81, CCG (Pro) to CTG(Leu), and exon 7, codon 121, ATA (Ile) to ATG (Met), in Trp53 inLY-2 SCC cells (data not shown). Thus, NF-�B appears to contributeto the overexpression of Trp53 in LY-2 SCC cells.

Nuclear Factor-�B–Related Motifs in the Promoter and Mes-senger RNA Coding Region of Genes Up- and Down-Regulated byNuclear Factor-�B in Squamous Cell Carcinoma. To examinewhether these and the larger list of genes modulated by I�B�M asdetected by microarray contain DNA sequence motifs that are putativetargets for NF-�B regulation, we performed a search of the DNA thatincluded 2 kb of the promoter region and the full-length sequence ofeach gene. As annotated in Tables 1 and 2, multiple genes wereidentified that contain sequences for NF-�B DNA binding motifs (�)and/or the ACTACAG 7-nucleotide motifs (#), recently shown tomediate posttranscriptional down-regulation of the genes Myo D andSox9 by NF-�B (13). Fully 105 of 167 (63%) of the genes classifiedas increased with tumor progression contained putative NF-�B pro-moter DNA binding sequences, approximating the percentage of�69% found to be down-modulated by I�B�M (see Table 1, GenesIncreased with Tumor Progression). These include cyclin D1, Trp53,and IAP-1 confirmed by Western and Northern blot analysis. Of thegenes decreased with tumor progression (Table 2), 47 of 141 (33%)also contained NF-�B DNA binding motifs, including �-catenin. TheDNA binding motif associated with p65 RelA (12) expressed in LY-2and other SCCs was present in 42% of the genes showing increasedexpression but in only 14% of the genes showing decreased expres-sion. Overall, NF-�B (63%) and p65 (42%) DNA binding motifs wereincreased in LY-2 cells with tumor progression when compared withNF-�B motifs detected in 25% of 900 genes randomly selected fromthe array list, indicating that the increase in representation of thesemotifs in genes expressed is unlikely to have occurred by chance(�2, P � 0.001).

The motif ACTACAG shown to be involved in posttranscriptionaldown-regulation of the genes MyoD and Sox9 by NF-�B (13) wasdetected in the promoter, exon, or intronic DNA sequence of over50% of genes exhibiting decreased expression with tumor progres-sion, but these motifs were present at a similar frequency in the DNAof genes showing increased expression and in 104 genes selectedrandomly and analyzed by the National Center for Bioinformatics (�2,P 0.05). The 7-mer motif was present in the coding portion of 30of 167 (17%) and 18 of 141 (13%) genes showing increased ordecreased expression, respectively. When only those genes that con-tain the 7-mer within the coding sequence and in-frame as found inMyoD and Sox9 (13) are included, we detected this motif in 14 of 167genes increased with tumor progression and 11 of 141 genes de-creased with tumor progression, a frequency of �8%.

Fig. 4 shows the alignment of the ACTACAG motif and adjacentDNA sequences for genes that showed conserved nucleotides withthose of MyoD and Sox9 and/or within the same region of their humanhomologues (�). Cytosines (C) located 5 and/or 8 nucleotides 5� andafter the 7-mer and C or G nucleotides (S) located 10, 13, 14, and 16nucleotides 3� to the motif were most common. Although thesehomologies suggest a possible relationship to the mRNA regulatorymotifs found in MyoD and Sox9, all but Atp11a lacked multiplerepeats, and Fn1 contained a motif not found in its human homologue.Thus, whereas the NF-�B DNA binding sequences involved in transcrip-tional regulation are significantly increased in genes differentially

expressed in SCC, the significance and functional role of ACTACAGmotifs found in the genes detected remain to be determined.

Effect of Inhibition of Nuclear Factor-�B on the MalignantPhenotype of Squamous Cell Carcinoma. The putative functionalrole of the validated genes and other genes in Tables 1 and 2 in cellproliferation and death, inflammation/angiogenesis, migration/metas-tasis, metabolism, stress, signaling, gene and protein expression, andstructure is consistent with the complex phenotypic differences be-tween keratinocytes and highly metastatic SCC cells. To establishwhether the modulation by NF-�B is associated with effects on themalignant phenotype, we examined the effect of modulation ofI�B�M on several features of malignancy in LY-2 cells. DOX-induced expression of I�B�M had minor effects on proliferationbeginning at day 3 as measured by MTT densitometric assay (Fig.5A), corresponding to the interval when DOX-induced inhibition ofNF-�B activation was detected (Fig. 1A). No changes in cell mor-phology, trypan blue uptake, or detachment were evident on micros-copy (data not shown). To determine whether the decreased densityobserved by day 5 was related to cell death, cell cultures wereexamined by microscopy and by DNA cytofluorometry for cell cyclechanges and subcellular DNA fragmentation that are produced on celldeath. Cells expressing I�B�M began to show blebbing, fragmenta-tion and detachment (data not shown). This was accompanied by theloss of DNA from G, S, and M phases and shift of �50% of the DNAto the sub–G0-G1 fraction, consistent with cell death and fragmenta-tion (Fig. 5B).

Because a number of genes potentially involved in cell adhesion,migration, and invasion were identified by array, we compared themigration and invasion of Pam 212 and LY-2 cells and examined theeffect of inhibition of NF-�B on migration and invasion through Matrigelmatrix in a two-chamber assay between 24 and 72 hours after addition ofDOX, before significant cell loss or death was detected. Fig. 6A showsthat control LY-2 cells � DOX exhibit a similar increase in invasion ofmatrix relative to Pam 212. However, the invasiveness of four of fourLY-2 clones conditionally expressing I�B�M was reduced to levels at orbelow those observed for Pam 212, suggesting that the increase in NF-�Bobserved in LY-2 cells plays a role in the increased migration andinvasiveness of these metastatic cells.

Because NF-�B regulates Gro-1, a factor that promotes tumorigen-esis and host angiogenesis (5, 14), the effect of inhibition of NF-�B byI�B�M on tumorigenesis and angiogenesis was examined. Fig. 6Bshows the tumor growth of control LY-2 cells and the LY-2 clone 24C

Fig. 4. Comparison of ACTACAG motifs and adjacent DNA sequences betweenMyoD, Sox9, and selected genes detected by array showing conserved nucleotides. Thecoding sequences of MyoD, Sox9, and a panel of selected genes were aligned withreference to the ACTACAG motif. Conserved nucleotides among the sequences areshaded. S represents C or G. Asterisk indicates that homologous nucleotides were presentin the same region in corresponding human sequences.

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before and after addition of DOX to the drinking water of congenicBALB/c SCID mice. On addition of DOX (arrow), the parental linecontinued to grow, whereas LY-2 24C tumor rapidly regressed withinhibition of NF-�B by I�B�M. No regrowth was detected in miceundergoing complete regression, but four mice retained small palpablenodules. To determine whether the inhibition was associated withdecreased angiogenesis, the density of CD31-expressing blood vesselsin the control and four mice bearing residual 24C tumors was com-pared. Fig. 6C illustrates the difference in tumor size in H&E-stainedspecimens and the associated difference in density of CD31 stainingvessels. The microvessel density was significantly reduced in 24Ctumors compared with control LY-2 tumors treated with DOX(53 � 15 versus 175 � 23, mean number of vessels per high-poweredfield � SD; n 4; P � 0.05). The clone transfected with I�B�Mgrew more slowly than the parental control, even in the absence ofDOX. This slower tumor formation was associated with a �10% to30% lower expression of NF-�B-modulated angiogenesis factorsGro-1 and vascular endothelial growth factor in 24C and other clonescompared with parental LY-2 cells, suggesting a degree of “leakiness”and weak suppression of NF-�B-inducible genes by the Tet-induciblepromoter, even in the absence of DOX (data not shown).

DISCUSSION

In this study, we obtained evidence for differential expression of adiversity of known NF-�B–related genes and provide evidence thatmodulation of the NF-�B pathway directly or indirectly modulates asignificant portion of these and other genes in the molecular profilealtered with malignant progression in a syngeneic model of SCC. Thearray profile obtained with the expanded 15,000-element microarray

reproduced differences in expression of a number of NF-�B–relatedgenes detected and validated by Northern blot in our previous study,as well as other genes reported to be differentially expressed andimportant in oncogenesis of SCC. We confirmed that I�B�M expres-sion suppressed NF-�B reporter and DNA binding activity and mod-ulated protein and mRNA expression of several putative NF-�B targetgenes differentially expressed in SCC, including cyclin D1, Trp53,IAP-1, and �-catenin (15–19). Bioinformatic analysis revealed thatthe sequence of these and many of the other putative NF-�B–modu-lated genes contain promoter or coding sequences homologous to theNF-�B DNA binding motif or an ACTACAG 7-nucleotide motifrecently shown to mediate posttranscriptional down-regulation byNF-�B of MyoD and Sox9 mRNA expression (13). Consistent withthe diversity and putative function of many of these genes, inhibitionof NF-�B was found to inhibit proliferation, cell survival, migration,angiogenesis, and tumorigenesis. These results provide evidence thatNF-�B is an important modulator of the gene expression profile andmalignant phenotype in SCC and provide an expanded list of candi-date genes potentially modulated directly or indirectly by NF-�B to beinvestigated in future studies.

Homologues of several of the genes detected with malignant pro-gression and modulated by NF-�B in this murine SCC model havebeen found to be differentially expressed and important in oncogen-esis in human SCCs. Cyclin D1 is overexpressed in the majority ofhuman head and neck squamous cell carcinoma (HNSCCs) and pro-motes proliferation and tumorigenesis (20). Cyclin D1 overexpressionresults from gene amplification in only a minority of SCCs, and theresults of this study provide evidence that NF-�B activation cancontribute to the overexpression of cyclin D1 in SCCs. The tumor

Fig. 5. Proliferation and DNA cell cycle analysis of LY-2P and LY-2 I�B�Mclones � DOX. A. MTT proliferation assay of LY-2P and LY-2 I�B�Mclones � DOX was performed as described in Materials and Methods, and theabsorbance on days 1, 3, and 5 was determined. Inhibition of proliferation wasobserved after DOX-induced expression of I�B�M, but not in control LY-2 cells.B. DNA cell cycle fluorescence analysis was performed on day 4 by flowcytofluorometry as described in Materials and Methods. A loss of G-S-M–phaseDNA and a marked increase in sub–G0-G1 DNA are observed in LY-2 23D and24C clones after DOX-induced expression of I�B�M but not in control LY-2Pcells.

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suppressor Trp53 is mutated and overexpressed in approximately halfof human HNSCCs and plays an important role in cell cycle arrest andDNA repair or apoptosis (21). The overexpression of Trp53 in LY-2and inhibition after inactivation of NF-�B suggest that NF-�B maycontribute to overexpression of Trp53. Increased expression of IAP-1(Birc2) was previously detected in metastatic SCC (4). The inhibitionof IAP-1 expression with inhibition of NF-�B in this study is con-sistent with the effect of inhibition of NF-�B in sensitizing LY-2 cellsto apoptosis and HNSCC to tumor necrosis factor-induced caspase-mediated cell death (22). Decreased expression of �-catenin has beenobserved and associated with metastasis of human oral SCC (23). Thelow expression of �-catenin in metastatic LY-2 cells relative tokeratinocytes and restoration of increased mRNA and protein levelsafter expression of I�B�M suggest that repression of �-catenin ex-pression can result from NF-�B activation in SCC. The effects ofmodulation of NF-�B on expression of cyclin D1, Trp53, IAP-1, and�-catenin observed in this study are consistent with those observedwith modulation of NF-�B in other malignancies (15–19).

When the expression of the four genes was compared by microarrayand Northern and Western blots, the direction of change for theproteins and genes studied was consistent for all four genes (Fig. 3Cand D). The percentage change was similar for protein and mRNA forcyclin D1 and �-catenin, whereas the percentage difference for pro-tein was greater than that observed for mRNA at the 72-hour time

interval for IAP-1 and p53. These differences between protein andmRNA could be due to a variety of mechanisms for individual genes.These include differences in kinetics related to other I�Bs (24),kinetics of modulation of RNA and protein, differences in stabilityand posttranscriptional processing of RNA, or differences in effi-ciency of translation. It will be important to determine whether dif-ferences among genes are due to different regulatory mechanisms infuture studies.

Bioinformatic analyses of the four validated genes for NF-�B–related motifs reveal that the sequences of the cyclin D1, Trp53,IAP-1, and �-catenin promoters contain NF-�B DNA binding motifs.The sequence of �-catenin also contains an ACTACAG 7-nucleotidemotif in the noncoding portion of the gene. The presence of this motifin the coding region has recently been shown to mediate posttran-scriptional down-regulation by NF-�B of MyoD and Sox9 mRNAexpression (13), but the role of the motif located in the noncodingportion of �-catenin remains to be determined. Although we haveobtained evidence that NF-�B modulates these and a wider profile ofgenes containing these motifs in Tables 1 and 2, it will be necessaryto determine individually for each gene by mutagenesis whether geneexpression is regulated directly by NF-�B or indirectly by othertranscription factors or regulatory molecules also modulated duringthe 72-hour interval required for significant DOX-induced expressionof I�B�M and inhibition of NF-�B.

Fig. 6. Effect of inhibition of NF-�B by I�B�Mon invasion, tumorigenesis, and angiogenesis. A,two-chamber invasion assay. Relative invasivenessas measured by absorbance of cells invadingthrough matrix to the lower chamber is correlatedwith the relative differences in metastatic potentialand NF-�B activation of transformed Pam 212,metastatic Pam LY-2, and LY-2P cells. The in-creased invasiveness of LY-2 I�B�M clones in theabsence of DOX is reduced after induction ofI�B�M. B. Tumor growth of LY-2 and LY-2 24Cin BALB/c SCID mice before and after addition ofDOX to drinking water. LY-2 24C grows moreslowly than LY-2 and regresses after induction ofI�B�M by DOX. C, comparison of histology andmicrovessel density of LY-2P and LY-2 24C tu-mors at week 10. H&E staining shows decreasedcellular density and size of LY-2 24C tumors rel-ative to LY-2P tumors. CD31 staining shows de-creased microvessel density of LY-2 24C tumorsrelative to LY-2P tumors.

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Gro-1, one of the genes consistently detected in LY-2 and aggres-sive SCC in this and previous studies, was also previously shown tobe modulated by NF-�B and to contribute to angiogenesis, tumori-genesis, and metastasis (5, 14). We showed that enforced expressionof Gro-1 in low-Gro-1–expressing Pam 212 cells converted thesecells to the LY phenotype and promoted increased angiogenesis,tumorigenesis, and metastasis, indicating that this NF-�B–regulatedgene contributes directly to the malignant phenotype of SCC in thismodel (14). Conversely, we show in this study that inhibition ofNF-�B and Gro-1 expression in LY-2 cells overexpressing Gro-1 isassociated with decreased angiogenesis and tumorigenesis. Humancytokine GRO1 and its homologue, interleukin-8, have been found tobe overexpressed in the majority of HNSCCs and many other cancers(3, 25). Consistent with the effects of inhibition of NF-�B by I�B� ongene expression and phenotype observed in this study, constitutiveexpression of an I�B� mutant in human HNSCC inhibited expressionof interleukin-8 and tumorigenesis of xenografts in mice (7). Althoughthe results suggest an important functional role of NF-�B in angio-genesis and tumorigenesis, the decrease in angiogenesis observedcould be due in part to apoptosis and reduction in tumor massproducing angiogenesis factors, as well as decreased transcription.

Our previous studies and the present molecular profiling study ofmalignant progression reveal differences in expression of a variety ofother putative NF-�B–modulated genes that are consistent with ex-pression patterns observed more broadly in SCC. Glutathione S-transferase was increased in LY-2 and inhibited with suppression ofNF-�B activation. Overexpression of glutathione S-transferases in-volved in metal metabolism has been detected and shown to beimportant in resistance of SCC to cis-platinum and other chemother-apy agents (26). Expression of the cell adhesion molecules integrin�3�1 and decreased expression of cadherin 1 and �-catenin protein areoften observed and associated with poor prognosis in SCC and otherepithelial cancers (2, 23). Decreased expression of the cell-cell recep-tor Ly-6 was also confirmed previously in LY SCC cells (27). Finally,the detection of several classes of molecules previously detected astumor specific and tumor-associated antigens in SCC such as Ly-6,integrins, DEAD-box helicases, and ribosomal components (2, 27–29)suggests that NF-�B may contribute to expression of antigenic mol-ecules detected by antibodies and T cells. Identification of NF-�B–regulated genes could help determine the identity of antigens forimmune therapy as well as their function in the malignant phenotype.The differences in expression of these candidates implicated in reg-ulation of resistance to chemotherapy-induced apoptosis, adhesion,and immune antigenicity in this model are therefore consistent withbroader expression in SCC and make the role of NF-�B in theirregulation of broader potential relevance.

We noted in the previous study with a first-generation 4,000-element murine array that a significant portion of the genes overex-pressed in multiple metastatic reisolates were reported to be related tothe NF-�B pathway. Consistent with this, a search of PubMed re-vealed that 40% of the up-regulated genes and �28% of the down-regulated genes detected in LY-2 with the 15,000-element array in thepresent study were related previously to NF-�B. The detection of bothknown and additional up- and down-regulated genes with modulationof NF-�B prompted us to undertake an analysis to determine whetherthe genes identified contained common regulatory motifs related toNF-�B. Bioinformatic analysis of the sequence of genes detected withtumor progression of LY-2 in this study revealed that 63% of thegenes increased in LY-2 cells contained NF-�B binding motifs in thepromoter region. Of the candidates showing lower expression, 33%contained NF-�B binding motifs, and �8% contained one or moremotifs found in MyoD and Sox9 mRNA implicated in NF-�B–medi-ated posttranscriptional down-regulation (13). The detection of se-

quences homologous to the 7-nucleotide motif detected in MyoD andSox9 mRNA suggests that this NF-�B–regulated mechanism may beinvolved in down-regulation of other genes besides MyoD. The res-toration of expression of these genes back up to the levels observed inkeratinocytes after expression of I�B�M in LY-2 provides support forthis hypothesis. The mechanism underlying NF-�B modulation ofMyoD and Sox9 mRNA and relevance to expression of other genes incancer remain to be established.

Cumulative evidence in head and neck, lymphoid, breast, gastric,colorectal, and prostate cancers is consistent with the hypothesis thatNF-�B is constitutively activated and a major “culprit” in the patho-genesis of cellular and host alterations in these cancers (reviewed inrefs. 25 and 30). NF-�B has been implicated in the oncogenesis of anumber of other malignancies, indicating that NF-�B activation maybe a common pathway of broad importance in cancer (30). Viral andcellular members of the NF-�B family have been shown to be trans-forming (31, 32), and oncogenic activation of RAS, ABL, LMP-1,interleukin-1, and epidermal growth factor receptor has been shown tocontribute to activation of NF-�B in different cancers (10, 33–36).Constitutive activation of NF-�B and NF-�B–inducible genes hasbeen detected in prostate and breast cancers, melanomas, and lym-phomas (25, 37–41). NF-�B has been implicated in promoting ex-pression of the phenotypic changes and genes involved in inhibition ofprogrammed and therapeutic cell death (18, 22, 41–44), proliferation(15), tumorigenesis (7, 25, 34), angiogenesis, invasiveness, and met-astatic potential (9, 25, 38).

Furthermore, molecular profiling has revealed that NF-�B mayregulate a diverse repertoire of genes in SCC (this study) and inHodgkin’s and non-Hodgkin’s lymphomas (39–41). We have pro-vided evidence that NF-�B modulates many of the genes and pheno-typic features differentially regulated during malignant progression inthis stepwise model of SCC. The broader significance of these resultsis independently supported by our earlier results showing that NF-�Bis activated in multiple murine and human SCCs (2, 5, 6) and thatconstitutive inhibition of NF-�B in human SCC inhibits cell survival,angiogenesis factor expression, and growth of xenografts (7, 9). Thegenes detected represent programs that are functionally involved inmany of the phenotypic features that constitute the hallmarks ofcancer (1). These results suggest that NF-�B is a key molecular switchof the alterations in genotype and phenotype in malignant progressionof SCC. Increasing appreciation of the role of NF-�B in regulation ofexpression of a diversity of genes in cancer is consistent with theevolutionarily importance of this transcription factor in regulatingprogrammed or stereotyped gene responses to injury and pathogens(25, 30).

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[CANCER RESEARCH 64, 8130–8133, November 1, 2004]

Corrections

FX Enzyme Controls the Adhesive Properties of CRC

In the article on how FX enzyme controls the adhesive properties ofCRC in the September 15, 2004 issue of Cancer Research (1), someof the labeling in Figures 7 and 10 was incorrect. The correctedfigures are below.

1. Zipin A, Israeli-Amit M, Meshel T, Sagi-Assif O, Yron I, Lifshitz V, Bacharach E,Smorodinsky NI, Many A, Czernilofsky PA, Morton DL, Witz IP: Tumor-microenviron-ment interactions: the fucose-generating FX enzyme controls adhesive properties of colo-rectal cancer cells. Cancer Res 2004;64:6571–8.

Chromosome 11q LOH in Human Breast Cancer

In the article on chromosome 11q LOH in human breast cancer inthe September 1, 1994 issue of Cancer Research (1), the name of oneof the contributing authors was misspelled. The correct spelling isRobert Winqvist.

1. Hampton GM, Mannermaa A, Winqvist R, Alavaikko M, Blanco G, Taskinen PJ,Kiviniemi H, Newsham I, Cavenee WK, Evans GA: Loss of heterozygosity in sporadichuman breast carcinoma: a common region between 11q22 and 11q23.3 Cancer Res1994;54:4586–9.

p110� Isoform of PI3 Kinase in Tumor Endothelium

In the article on p110� Isoform of PI3 Kinase in Tumor Endothe-lium in the July 15, 2004 issue of Cancer Research (1), the name ofone of the contributing authors, Jeffrey Brousal, was missing. Thecorrect list of authors should read: Ling Geng, Jiahuai Tan, EricHimmelfarb, Aaron Schueneman, Ken Niermann, Jeffrey Brousal,Allie Fu, Kyle Cuneo, Edward A. Kesicki, Jennifer Treiberg, Joel S.Hayflick, and Dennis E. Hallahan. Dr. Brousal’s affiliation is theDepartment of Radiation Oncology, Vanderbilt University School ofMedicine, Nashville, Tennessee.

1. Geng L, Tan J, Himmelfarb E, Schueneman A, Niermann K, Brousal J, Fu A, Cuneo K,Kesicki EA, Treiberg J, Hayflick JS, Hallahan DE. A specific antagonist of the p110�catalytic component of phosphatidylinositol 3�-kinase, IC486068, enhances radiation-induced tumor vascular destruction. Cancer Res 2004;64:4893–9.

Glioblastoma-Founding Human Neural Precursors

In the article on glioblastoma-founding human neural precursors inthe October 1, 2004 issue of Cancer Research (1), the e-mail addressof R. Galli should have been included in the requests for reprintssection. Dr. Galli’s e-mail address is [email protected].

1. Galli R, Binda E, Orfanelli U, Cipelletti B, Gritti A, De Vitis S, Fiocco R, Foroni C, DimecoF, Vescovi A. Isolation and characterization of tumorigenic, stem-like neural precursorsfrom human glioblastoma. Cancer Res 2004;64:7011–21.

NF-�B in Squamous Cell Carcinoma

In the article on NF-�B in squamous cell carcinoma in the Sep-tember 15, 2004 issue of Cancer Research (1), the entries in Tables 1and 2 indicating NF-�B modulated genes should have been boldfaced.The corrected Tables 1 and 2 are reproduced below.

1. Loercher A, Lee TL, Ricker JL, Howard A, Geoghegen J, Chen Z, Sunwoo JB,Sitcheran R, Chuang EY, Mitchell JB, Baldwin AS Jr, Van Waes C: Nuclear factor-�Bis an important modulator of the altered gene expression profile and malignantphenotype in squamous cell carcinoma. Cancer Res 2004;64:6511–23.

Fig. 7. Expression of FX mRNA, FX protein and of sLe-a by SW620 cells stablytransfected with FX siRNA. A, FX mRNA. Expression was determined by Northern blotanalysis. Values represent the ratio between the signal of FX mRNA in the cells and thesignal of 18S rRNA in the same cell sample. B, FX protein. Expression was determinedas in Fig. 2A. C, sLe-a. Expression was determined as in Fig. 2B. A representativeexperiment (of three performed) is presented (360 cells � control cells; 205 cells � FXsiRNA transfectants). M � mean fluorescence; %pos � % positive cells.

Fig. 10. Expression of fucosylated proteins by SW620 cells stably transfected with FXsiRNA. Lysates of control (360) and FX siRNA (205) transfected cells were assayed forthe expression of fucosylated proteins by Western blot analysis using horseradish perox-idase-conjugated Ulex europaeus agglutinin 1. Expression of FX protein in the two cellpopulations was determined as in Fig. 2A. Glyceraldehyde-3-phosphate dehydrogenase(GAPDH) served as loading control.

8130

Table 1 Selected list of genes increased with tumor progression

FunctionGene Symbol Clone ID NF-�B Association

Fold change

LY-2/Ker LY-2/IkB-aM

Cell cycle/growthCyclin D1* Ccnd1 H3084D05 Target gene 3.351 �2.266Cyclin D2* Ccnd2 H3152D01 Target gene 2.957 �2.05Growth arrest specific 5* Gas5 H3113A12 6.304 �7.813Milk fat globule-EGF factor 8* Mfge8 H3126F11 Target gene 2.594 �3.525Protein phosphatase 3*† Ppp3cb H3065C08 Inhibitor of NF-�B 3.117 �2.231Proliferating cell nuclear antigen* Pcna H3021F12 Target gene 5.775 �2.811

ApoptosisBaculoviral IAP repeat* Birc2 H3074A02 Target gene 9.154 �6.013Bcl-2 related ovarian killer*† Bok1 H3081D02 1.994 �3.461Immediate early response 3* Ier3 H3057B07 Target gene 2.865 �3.851Transformation related protein*† Trp53 H3142D07 Target gene 2.9 �4.142Uchrp* Uchrp IMAGE:605056 2.188 �4.353

Inflammation/angiogenesisColony stimulating factor 1* Csf1 H3057D05 Target gene 14.639 �5.574Complement component 3*† C3 H3054A08 Target gene 5.145 �8.303FGF receptor* Fgfr4 IMAGE:406823 Target gene 1.982 �1.991Gro 1 oncogene* Gro1 H3051F10 Target gene 12.394 �4.094Histocompatibility 2-L* H2-L H3096A12 Target gene 2.342 �3.431Histocompatibility 2-D* H2-D H3141B11 Target gene 2.968 �3.927Interferon receptor* Ifnar H3118F09 Inhibitor of NF-�B 4.5 �3.054Lymphocyte antigen complex* Ly6e H3027D05 Inducer of NF-�B 4.029 �2.613

MetastasisIntegrin � 3*† Itga3 H3137A03 Inducer of NF-�B 15.597 �3.917Laminin � 5*† Lama5 H3002G01 Target gene 2.272 �2.967Laminin receptor 1 Lamr1 H3075G08 2.169 �1.619Plasminogen activator, tissue Plat H3080H11 Target gene 2.667 �2.487Procollagen type 5 � 2 Col5a2 H3156E09 Target gene 5.103 �2.094Syndecan 1* Sdc1 H3013F05 Target gene 2.948 �2.034

MetabolismATPase H� transport*† Atp6b H3120H04 Inhibitor of NF-�B 2.247 �2.858Branched chain ketoacid dehyd* Bckdk H3136B09 2.455 �3.042Choline kinase*† Chk H3088E07 Inducer of NF-�B 6.662 �3.252Cytochrome p450* Cyp1b1 J0216F07 Target gene 22.289 �4.286Glutathione-S-transferase* Gstm1 H3133A06 Target gene 3.037 �3.061Low density lipoprotein receptor* Ldlr H3014C04 Target gene 2.689 �3.226Mannose-6-phosphate receptor† M6pr H3092C05 Inhibitor of NF-�B 6.303 �3.612Potassium intermediate* Kcnn4 H3054H04 2.778 �2.197Solute carrier family 12*† Slc12a2 H3077B02 2.551 �2.145

Stress responseHeat shock protein, 70 kDa* Hspa5 H3032A08 Activates NF-�B 14.303 �6.369Heat shock protein 84 kDa* Hsp84 H3042G07 Activates NF-�B 3.437 �7.198Heat shock protein 86 kDa* Hsp86 H3023G01 Activates NF-�B 2.61 �2.34Heat shock protein cognate 70* Hsc70 H3133H01 Binds NF-�B 3.42 �10.229Superoxide dismutase* Sod1 H3130B11 Target gene 4.784 �2.145

Signal transductionAXL receptortyrosine kinase*† Axl H3152F05 Inhibitor of NF-�B 2.459 0.938CD97 (EGF-TM7)*† Cd97 H3032G06 2.283 �1.352Interleukin-1 receptor associated* Il1rak H3042E08 Activates NF-�B 1.999 �1.296Frizzled 7 homolog Fzd7 H3031A03 2.717 �0.843Growth arrest & DNA damage specific* Gadd45g H3054C02 Activates NF-�B 3.144 �1.407Growth factor receptor bound* Grb2 H3153D02 Activates NF-�B 2.483 �2.341N-myc downstream regulated* Ndr2 G0110H06 Inhibits p50 2.248 �2.039P13 kinase regulatory* Pik3r1 H3067B08 Activates NF-�B 4.206 �3.539Protein tyrosine phosphatase*† Ptpn13 H3118G02 Inhibitor of NF-�B 2.844 �9.071Ras p21 protein activator 3*† Rasa3 H3054E01 Activates NF-�B 3.223 �2.71Ras-related C3* Rac1 IMAGE:477981 2.042 �3.875Transferrin receptor* Trfr H3059G03 2.216 �1.145

Nuclear proteins/transcription factorsActivating transcription factor† Atf2 J0221F08 1.606 �2.696Breast cancer, early onset† Brca2 H3069F08 2.216 �0.604Butyrate response factor* Brf2 H3015E08 2.072 �2.008High mobility group AT* Hmga1 H3029B11 Target gene 2.816 �2.591Jerky* Jrk H3119F06 2.849 �3.329Myelocytomatosis oncogene* Myc H3089H11 Target gene 2.224 �2.42Nuclear factor �B p105* Nfkb1 H3072E09 2.919 �1.443Sex comb on midleg-like 1† Scml1 H3113B01 2.355 �1.154Yes-associated protein 65 kDa Yap H3089H07 2.072 �1.746

RNA processingDEAD box protein 3* Ddx3 H3018F11 2.976 �2.055DJ-1 protein† DJ-1 H3150D06 3.155 �4.611FGF inducible 14 Fin14 H3018G01 2.358 �3.355Nuclear ribonuclease Hnrpa1 H3111H11 4.926 �2.785RNA polymerase 1-1* Rpo1-1 H3049D09 2.703 �2.328

Protein synthesis/modificationERO1 like*† Ero1l H3126B01 2.529 �2.456Ribosomal protein L27a Rpl27a H3009B05 2.183 1.369

8131

Table 1 Continued

FunctionGene Symbol Clone ID NF-�B Association

Fold change

LY-2/Ker LY-2/IkB-aM

Ribosomal protein L8 Rpl8 H3141F09 2.778 1.005Ribosomal protein S18* Rps18 H3006C11 2.487 �2.424Ubiquitin activating enzyme E1 Ube1x H3022E03 Phosphorylates �B 5.313 �3.139Ubiquitin B Ubb H3138A08 Labels �B 8.144 �1.171Ubiquitin conjugating enzyme E2 Ube2h H3057B09 Phosphorylates �B 3.333 �1.199Ubiquitin conjugating enzymeE3†

Ube3a H3102B01 Phosphorylates �B 4.878 �5.096

Ubiquitin specific protease 9† Usp9x H3139F12 3.769 �3.175Structural proteins

Alpha tropomyosin* Tpm1 H3120G06 Binds p65 2.421 �4.36Cadherin* Cdh3 H3018F05 Inflammatory 2.328 �2.706Capping protein � 2 Cappa2 H3085F12 14.293 �7.458Dystroglycan 1* Dag1 H3008B05 Activates NF-�B 2.593 �4.738Epithelial protein lost*† Eplin H3153C05 Activates NF-�B 2.066 �2.971Fascin homolog 1* Fscn1 H3006D08 2.315 �2.581Four and a half LIM domains* Fhl2 H3033C07 2.169 �1.189Keratin complex 1 acidic* Krt1-18 H3021B02 Target gene 3.105 �5.517Keratin complex 2 basic* Krt2-8 H3031C01 2.094 �5.568PDZ and LIM domain 1* Pdlim1 J0824B03 5.7 �3.775Protocadherin 7*† Pcdh7 H3067F12 Inflammatory 6.556 �1.026Thymopoietin* Tmpo H3096B08 9.939 �8.153

OtherGlobin inducing factor† Gbif H3053F12 2.003 1.002Metallothionein 2 Mt2 H3013D11 Inhibits I�B degradation 2.145 �3.029Next to the Brca1*† Nbr1 H3061D04 2.367 �1.888RAN binding protein* Ranbp9 H3013A10 Accumulates I�B� 2.87 �3.189Repeat family 3 gene* Llrep3 H3107F07 3.556 �7.704Ring finger protein 19* Rnf19 H3153A08 Activates NF-�B 2.148 �4.402Sema domain, immunoglobin*† Sema3f H3134D09 2.191 �1.667Suppressor of Lec15† Supl15h H3090D12 2.001 �1.383TGF � inducible transcript* Tgfb1i1 H3122H01 3.068 �2.974

NOTE. Total number of genes regulated by NF-�B � 105/167. Total number of genes previously associated with NF-�B � 67/167.* Genes containing �B site in promoter region.† Genes containing ACTACAG motif in coding sequence.

8132

Table 2 Selected list of genes decreased with tumor progression


Fold change

LY2/Ker LY2/IkB-aM

Cell cycle/growthC-src tyrosine kinase* Csk L0237H04 �2.693 2.358Calmodulin Calm H3006H05 Activates NF-�B via IKK �2.554 2.42Cell division cycle homolog 25a Cdc25a H3050E04 �2.341 2.269Cell division cycle homolog 45 Cdc45l H3003E07 �2.032 2.029Cyclin C Ccnc C0117F09 �2.739 2.418Cyclin E2* Ccne2 C0186A01 �2.309 2.155Cyclin dependent kinase 4 Cdk4 H3147D06 Target gene of NF-�B �2.734 2.05Cyclin dependent kinase inhibit* Cdkn1c H3097D03 �5.208 2.913Platelet derived growth factor* Pdgfa H3146C02 �2.15 2.906

ApoptosisATP binding cassette* Abcd3 H3143E03 NF-�B site in promoter �2.89 2.535Bcl2/adenovirus E1B Bnip3 H3103B07 Transient inhibitor of NF-�B �2.364 2.436Fas associating w/death domain Fadd H3095D08 Inducer of NF-�B �3.597 2.975

Inflammation/angiogenesisCoagulation factor III*† F3 H3014G02 Activates NF-�B via IKK �4.672 3.858Interleukin 17 receptor Il17r H3008A03 Activates NF-�B via MAPK �3.021 3.048Interleukin 2 receptor Il2ra J0052C08 NF-�B site in promoter �2.262 2.207Lymphocyte antigen 6 complex Ly6 H3115A08 Inducer of NF-�B �5.617 3.311Prothymosin � Ptmb4 H3143A02 �21.739 8.858

MetastasisA disintegrin/MMP Adamts1 H3034B07 �2.695 2.736Cadherin 1* Cdh1 H3076B06 Associated with inflammation �2.597 2.313Kangai 1† Kai1 H3154D02 Target gene of NF-�B �2.816 2.139Lipocalin 2 Lcn2 H3083G02 Associated with inflammation �2.506 11.235Procollagen type 1 � Col1a2 H3125D01 �4.901 2.886Procollagen type II � Col2a1 H3026G09 �6.896 2.139Procollagen type III � Col3a1 H3005D11 Inducer of NF-�B �3.205 4.268Secreted acidic C-rich Sparc H3026D08 �8.928 2.872Tissue inhibitor of MMPs Timp3 H3031E01 Two NF-�B sites in promoter �3.3 3.289

MetabolismATPase, type 11A† Atp11a H3097B05 �2.888 2.734ATP synthase H� transport Atp5j2 H3118C01 Inhibitor of NF-�B �2.191 2.111Glutathione peroxidase* Gpx3 J0088G08 Inhibitor of NF-�B upregulates IkBa normal half-life �2.604 4.065Lipopolysaccharide binding*† Lbp H3086G08 Activates NF-�B via MAPK �2.977 2.103Phosphoprotein enriched† Pea15 H3014G07 Inducer of NF-�B �2.424 1.159Pyruvate dehydrogenase*† Pdha1 H3068G07 NF-�B site in promoter �3.021 1.643Sterol carrier protein 2*† Scp2 H3122F12 �2.412 1.488

Stress responseCrystallin � 2 Crya2 H3143B04 Inhibitor NF-�B �3.921 3.479

Signal transductionAdenylate kinase† Ak2 H3052D11 �3.755 3.546Max dimerization protein 4*† Mad4 H3131B07 �2.008 2.131MAD homolog 4 Madh4 H3128C04 �2.765 2.114NF-�B enhancer inhibitor* Nfkbia H3026A08 �1.433 4.611NIK-related kinase Nrk H3008B02 Activates NF-�B �2.244 4.859Phosphoglycerate kinase* Pgk1 H3023D06 �2.659 2.061Protein tyrosine phosphatase 4 Ptp4a2 H3088F03 �2.118 1.615Rho-associated coiled-coil Rock1 H3069C09 �2.808 3.183TNF receptor associated factor Traf1 H3015E06 NF-�B dependent �2.011 1.243

Nuclear proteins/transcription factorsCbp/p300 interacting transactivation Cited4 H3076H08 NF-�B co-activator �2.178 2.745High mobility group box 1 Hmgb1 H3126A05 Binds p50 subunit �3.104 1.739Jun oncogene Jun H3058C09 NF-�B co-activator �2.906 2.057Ras-related C3 Rac1 H3018C09 Inducer of NF-�B �2.004 3.329

RNA processingNuclear protein 220† Np220 H3029A07 �2.259 1.827RNA polymerase II* Rpo2-3 H3055H08 Coactivator of p65 �2.639 4.501

Protein synthesis/modificationEukaryotic translation 4g2 Eif4g2 H3113E10 �4.698 2.222Nedd4 WW-binding protein 4* N4wbp4 H3062G06 �6.966 2.814

Structural proteinsAlpha 2 glycoprotein 1*† Azgp1 IMAGE:521249 �2.013 1.398Beta spectrin 2*† Spnb2 H3010G09 �3.781 1.015Catenin beta* Catnb H3031E05 Regulated by IKK �3.998 3.061Fibronectin† Fn1 H3116A10 NF-�B site in promoter �6.201 4.723Catenin � 1*† Catna1 H3018E08 �1.996 1.723Tenascin C Tnc L0062E01 NF-�B site in promoter �2.557 1.956

OtherDeleted in polyposis Dp1 J0420H06 �2.473 2.137Insulin-like growth factor r2 Igfr2 H3148G08 �2.765 2.494Ninjurin 1* Ninj1 H3072B10 �2.579 4.878Rabaptin 5*† Rab5ep H3002C01 �2.739 1.333Topoisomerase II �*† Top2a H3139A05 Inducer of NF-�B �2.427 2.893Tumor differentially expressed† Tde11 H3014H10 �2.593 1.18WW domain binding 5* Wbp5 H3127H02 �2.087 3.313Zinc finger protein 68*† Zfp68 H3058F07 �4.122 5.885

NOTE. Number of genes regulated by NF-�B � 47/141. Number of genes previously associated with NF-�B � 39/141.* Genes containing kB site in promoter region.† Genes containing ACTACAG motif in coding sequence.

8133

2004;64:6511-6523. Cancer Res Amy Loercher, Tin Lap Lee, Justin L. Ricker, et al. Squamous Cell CarcinomaGene Expression Profile and Malignant Phenotype in

B is an Important Modulator of the AlteredκNuclear Factor-

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